New Tricholidic Acid Triterpenoids from the Mushroom Tricholoma ustaloides Collected in an Italian Beech Wood

The secondary metabolites produced by Tricholoma ustaloides Romagn., a mushroom species belonging to the large Tricholoma genus (Basidiomycota, Tricholomataceae), are unknown. Therefore, encouraged by the interesting results obtained in our previous chemical analyses of a few Tricholoma species collected in Italian woods, we aimed to investigate the secondary metabolites of Tricholoma ustaloides. The chemical analysis involved the isolation and characterization of secondary metabolites through an extensive chromatographic study. The structures of isolated metabolites, including the absolute configuration, were established based on a detailed analysis of MS, NMR spectroscopic, optical rotation, and circular dicroism data, and on comparison with those of related compounds reported in the literature. Two novel lanostane triterpenoids, named tricholidic acids B and C, together with triglycerides, a mixture of free fatty acids, five unidentified metabolites, and the known rare saponaceolides F and J, tricholidic acid, and tricholomenyn C, were isolated from an EtOAc extract of fruiting bodies of Tricholoma ustaloides that were collected in an Italian beech wood. This is the second example of isolation of tricholidic acid derivatives from a natural source. Saponaceolides F and J exhibited high cytotoxicity (IC50 values ≤ 10 μM) against a panel of five human cancer cell lines. The toxicity against myeloid leukemia (HL-60), lung cancer (A-549), hepatocellular cancer (HepG2), renal cancer (Caki-1), and breast cancer (MCF-7) cells was higher than that shown by the very well-known cytotoxic drug cisplatin.


Introduction
The family Tricholomataceae, of the order Agaricales (Basidiomycota), comprises numerous genera. Among them, the genus Tricholoma (Fr.) Staude [1], including more than 250 species, is the largest one. All the known species are gilled mushrooms, mainly ectomycorrhizal with trees in the Pinaceae, Betulaceae, and Fagaceae families. They have a worldwide distribution [2] and are mainly found in temperate and subtropical zones in both the southern and northern hemispheres. The center of species richness appears to be in North America, from where more than 100 species have been reported [3], while 63 to 88 species are listed from Europe [4][5][6]. Several species are also described or reported from China, Japan, New Zealand, and Australia.
The taxonomy and phylogeny of the genus Tricholoma have been based, up to recent times, mainly on fungal morphological characters; however, the use of molecular methods based on nuc rDNA internal transcribed spacer ITS1-5.8S-ITS2 (ITS) sequences is becoming more and more important to analyze the diversity and distribution of several species [7][8][9]. Thus, phylogenetic analyses using nuclear and mitochondrial gene sequences have often proved that mushrooms collected with the same name in different places of the same country/continent [10,11] or in distant continents [8,10] must instead be placed Our phytochemical investigation of an aqueous methanol sub-extract of an EtOAc extract of T. ustaloides fruiting bodies led to the isolation of two C-30 terpenoids, saponaceolides J (1) [29] and F (2) [20,30], three cyclic lactone-containing lanostane triterpenoids, tricholidic acid (5) [31] and tricholidic acids B (6) and C (7), together with tricholomenyn C (8) [24] (Figure 1), mixtures of triglycerides and free fatty acids, and five unidentified compounds. Triterpenoids 6 and 7 are novel compounds. The structures with absolute configurations were established by extensive MS, 1D, and 2D NMR spectroscopic methods, as well as optical rotation and CD data and comparison with the literature. Moreover, the cytotoxic activities of compounds 1, 2, 5-7 were evaluated against myeloid leukemia (HL-60), lung cancer (A-549), hepatocellular cancer (HepG2), renal cancer (Caki-1), and breast cancer (MCF-7) cells. This paper reports the isolation, structural elucidation, and cytotoxicity of the isolates.≤

Mushroom Extraction and Isolation of Secondary Metabolites
Extraction of fruiting bodies has always been a critical step in the isolation of fungal metabolites because an incorrect procedure may readily lead to the formation of artifacts. In fact, enzymatic reactions and/or the alcoholic solvent often used for the extraction may cause extensive compound degradation [32]. To avoid these problems, according to an optimized procedure, freshly collected fruiting bodies of T. ustaloides were frozen at −20 °C, chopped still frozen, and extracted with EtOAc in the cold. After biomass separation by filtration, the solution was taken to dryness by solvent removal under a vacuum at ≤35 °C. Evaporation of the last part of the extract was facilitated by multiple additions of hexane. This expedient also prevented a dangerous concentration of AcOH in the extract. Acetic acid was inevitably produced by the contact of EtOAc with the large amount of H2O (about 90% w/w) contained in the fruiting bodies. The residue was then partitioned between hexane and MeOH-H2O, 9:1. This procedure led to the almost quantitative extraction of triglycerides (NMR) and other non-polar compounds by the hydrocarbon phase. The residue from the aqueous methanolic layer was finally fractioned in the standard way by multiple open air and medium pressure liquid chromatographic (MPLC) separations on silica gel and reversed-phase columns, to give pure (NMR and TLC) compounds 1, 2, 5-7, five unidentified metabolites A1-A5, and a mixture of common free Our phytochemical investigation of an aqueous methanol sub-extract of an EtOAc extract of T. ustaloides fruiting bodies led to the isolation of two C-30 terpenoids, saponaceolides J (1) [29] and F (2) [20,30], three cyclic lactone-containing lanostane triterpenoids, tricholidic acid (5) [31] and tricholidic acids B (6) and C (7), together with tricholomenyn C (8) [24] (Figure 1), mixtures of triglycerides and free fatty acids, and five unidentified compounds. Triterpenoids 6 and 7 are novel compounds. The structures with absolute configurations were established by extensive MS, 1D, and 2D NMR spectroscopic methods, as well as optical rotation and CD data and comparison with the literature. Moreover, the cytotoxic activities of compounds 1, 2, 5-7 were evaluated against myeloid leukemia (HL-60), lung cancer (A-549), hepatocellular cancer (HepG2), renal cancer (Caki-1), and breast cancer (MCF-7) cells. This paper reports the isolation, structural elucidation, and cytotoxicity of the isolates.

Mushroom Extraction and Isolation of Secondary Metabolites
Extraction of fruiting bodies has always been a critical step in the isolation of fungal metabolites because an incorrect procedure may readily lead to the formation of artifacts. In fact, enzymatic reactions and/or the alcoholic solvent often used for the extraction may cause extensive compound degradation [32]. To avoid these problems, according to an optimized procedure, freshly collected fruiting bodies of T. ustaloides were frozen at −20 • C, chopped still frozen, and extracted with EtOAc in the cold. After biomass separation by filtration, the solution was taken to dryness by solvent removal under a vacuum at ≤35 • C. Evaporation of the last part of the extract was facilitated by multiple additions of hexane. This expedient also prevented a dangerous concentration of AcOH in the extract. Acetic acid was inevitably produced by the contact of EtOAc with the large amount of H 2 O (about 90% w/w) contained in the fruiting bodies. The residue was then partitioned between hexane and MeOH-H 2 O, 9:1. This procedure led to the almost quantitative extraction of triglycerides (NMR) and other non-polar compounds by the hydrocarbon phase. The residue from the aqueous methanolic layer was finally fractioned in the standard way by multiple open air and medium pressure liquid chromatographic (MPLC) separations on silica gel and reversed-phase columns, to give pure (NMR and TLC) compounds 1, 2, 5-7, five unidentified metabolites A1-A5, and a mixture of common free saturated and

Structure Determination
Compound 1 was isolated as a white powder. The counting of protons and carbons occurring in the NMR spectra, together with the ion [M+NH4] + at m/z 518 (C30H48NO6 + ) in the DCI-MS (NH3) spectrum, indicated that the molecular formula was C30H44O6. The 1 H and 13 C NMR data of the tricyclic spiroacetal unit and the sesquiterpenoid moiety from C1 to C15 of compound 1 were almost identical to those of saponaceolide B (3) [19,30] [19,33]. Thus, the structure of compound 1 (Figure 2) is that of saponaceolide J, previously isolated from T. terreum [29]. The NMR data and the optical rotation of 1 corresponded with those in the literature [29]. Scheme 1. Extraction and isolation of secondary metabolites from Tricholoma ustaloides.

Structure Determination
Compound 1 was isolated as a white powder. The counting of protons and carbons occurring in the NMR spectra, together with the ion [M+NH 4 ] + at m/z 518 (C 30 H 48 NO 6 + ) in the DCI-MS (NH 3 ) spectrum, indicated that the molecular formula was C 30 H 44 O 6 . The 1 H and 13 C NMR data of the tricyclic spiroacetal unit and the sesquiterpenoid moiety from C1 to C15 of compound 1 were almost identical to those of saponaceolide B (3) [19,30] with the exception of the signals characteristic of an additional trisubstituted double bond [δ C 134.4 (s, C9 ) and 123.0 (d, C10 ), and δ H 5. 16 (1 H, t, J = 7.4 Hz, H9 )]. The HMBC spectrum suggested that the double bond was located between C9 and C10 based on the correlations of H15 a (δ H 4.45, 1 H, d, J = 13.5) and H15 b (δ H 4.28, 1 H, d, J = 13.5) with C9 , and those of H8 a (δ H 2.11, 1 H, m) and H8 b (δ H 2.02, 1 H, m) with C9 . Moreover, the presence of a double bond between C9 and C10 resulted in the upfield shift of C15 (δ C 60.0, t) and a downfield shift of C8 (δ C 27.7, t), compared to the corresponding signals in the 13 C NMR spectrum of the dihydroderivative, saponaceolide B (3) [19,33]. Thus, the structure of compound 1 (Figure 2) is that of saponaceolide J, previously isolated from T. terreum [29]. The NMR data and the optical rotation of 1 corresponded with those in the literature [29]. Compound 2 was isolated as a white powder. The counting of protons and carbons occurring in the NMR spectra, together with the ion [M + NH4] + at m/z 534 (C30H48NO7 + ) in the DCI-MS (NH3) spectrum, indicated that the molecular formula was C30H44O7, differing from compound 1 by one oxygen atom. The 1 H-NMR and 13 C spectra of 2 exhibited the same features as saponaceolide A (4) [18], including the presence of a hydroxy-substituted γ-lactone, which was indicated by the signals at δH 5.05 ppm (1 H, br d, J = 5.5 Hz, H10) and δC 66.3 (d, C10). The main differences between the NMR spectra of compounds 2 and 4 were the appearance in the spectra of 2 of an additional olefinic proton at δH 5. 16 (1 H, t, J = 7.0 Hz, H9′) and an additional double bond [δC 134.7 (s, C9′) and 123.0 (d, C10′)]. These data indicated the presence of a double bond between the carbons C9′ and C10′ of saponaceolide 2, as in saponaceolide J (1). Hence, the structure of compound 2 was determined to be saponaceolide F ( Figure 2). The NMR data and the optical rotation of 2 nicely corresponded to those of the metabolite previously isolated from T. saponaceum [20,30], although different solvents were used for the measurements [30].
Compound 5 was isolated as a crystalline compound, mp 208-212 • C, [α] D 23 = −128.1 (c = 3.6 mg/mL, CH 2 Cl 2 ). The formula C 32  (1 H, J = 2.5 Hz), assignable, through a 3 J correlation, to proton H3 geminal to an acetoxy group; and a septuplet (1 H, J = 1.2 Hz) at δ H 6.12, attributable to an olefinic proton (H24), that was coupled to the vinylic methyls H 3 (Table 1), which were assigned, respectively, to the carbonyls of a γ-lactone (C21), a carboxylic acid (C30), and an acetate group (C31). The IR bands at 1750, 1730-1700, 1690, and 1626 cm −1 sustained the presence of these functional groups, and the carboxylic function was fully confirmed by the formation of a methyl ester (δ H 3.70, 3 H, s, OMe) upon exposure of 5 to CH 2 N 2 . Other characteristic signals in the 13 C NMR spectrum of compound 5 appeared at δ C 83.7 (d, C16) and 77.4 (d, C3), 127.3 (0, C8) and 142.0 (0, C9), 122.9 (d, C24) and δ C 157.2 (s, C25). They were assigned, respectively, to a lactone and an acetoxy methine carbon, a tetrasubstituted olefin carbon, and a trisubstituted olefin carbon conjugated to the ketone CO (δ C 196.9, s, C23). Moreover, H 2 15 showed a 3 J correlation with the carboxylic carbon (δ C 177.6, s, C30), whereas H16 (δ H 5.15) and H20 (δ H 3.46) exhibited a 3 J and a 2 J correlation, respectively, with the γ-lactone carbonyl carbon (δ C 178.6, s, C21).  3 , δ H in ppm, J in Hz) and 13 C NMR (75 MHz, CDCl 3 , δ C in ppm) spectroscopic data of. compounds 5-7. These data clearly suggested that compound 5 was a pentacyclic lactonic acid lanostane triterpenoid, bearing an α-oriented acetoxy group at C3. In fact, the almost identical physical and spectroscopic data of compound 5 with the literature led us to identify compound 5 with tricholidic acid (Figure 2), whose structure was firmly established by an X-ray crystallographic analysis [31]. Moreover, the negative sign of the CD curve of 5 (∆e = −2.08 at about 225 nm), which was attributed to the lactone ring [31], indicated the same absolute configuration as tricholidic acid [31] (Figure 2 ] showed no coupling with H20. This finding suggested the presence of a substituent at C20. The strong band of chelated hydroxy groups in the IR spectrum of compound 6 and the occurrence of a quaternary carbon at δ c 76.6 (s) in the 13 C NMR spectrum of 6 clearly indicated the presence of an OH group at C20. Accordingly, due to the β-effect, both the C17 and C22 signals in the 13 C NMR spectrum of compound 6 were significantly shifted downfield in comparison with the corresponding signals in the spectrum of 5. Thus, the structure of 20-hydroxytricholidic acid was attributed to 6, which was named tricholidic acid B. The almost identical NMR data of compounds 5 and 6 from C1 to C14 (Table 1) indicated that they had the same relative configuration 3R*, 5R*, 10S*, 13R*, 14S*. About the relative configuration at C16, C17, and C20 stereocenters, the cis relationship between H16 and H17 was suggested by the vicinal coupling constant J 16,17 of 6.3 Hz. On the other hand, H16 was shifted downfield by about 0.3 ppm in the 13 H NMR spectrum of 6, compared to the corresponding signal of 5, whereas selective irradiation of H 3 18 produced a nOe effect on the signals of H 2 22. These data clearly indicated that the 20-OH group was cis to H16 and H17 and anti to H 3 18, suggesting the relative configuration 16R*, 17S*, 20R* ( Figure 2). Finally, the negative sign of the ECD curve, like that of 5, suggested the absolute configuration of 6 to be 3R, 5R, 10S, 13R, 14S, 16S, 17R, 20R.
Compound 7 was isolated as a sticky, pale yellow oil. It showed negative signs of the rotation and the Cotton effect; however, the values of [α] D 22 and ∆ε could not be determined because of the paucity of material. The MW = 526 and the molecular formula C 32 H 46 O 6 were established by the pseudomolecular ion [M+NH 4 ] + at m/z 544 (C 32 H 50 NO 6 + ) in the DCI-MS (NH 3 ) spectrum and the counting of protons and carbons occurring in the NMR spectra ( Table 1). The IR spectrum revealed the presence of γ-lactone (1770 cm −1 ), ester (1730 cm −1 ), and carboxylic (1700 cm −1 ) groups. These functions gave rise to three singlets at δ c 178.7, 170.6, and 177.2, respectively, in the 13 C NMR spectrum of 7. Moreover, the acid 7 afforded a methyl ester (δ H 3.68, 3H, s, OMe) on treatment with CH 2 N 2 . With the aid of 1 H, DEPT and HSQC spectra, the 46 proton and the 32 carbon resonances as displayed by the 1 H NMR and 13 C NMR spectra of 7 (Table 1)  Thus, the structure of 23-deoxotriholodic acid was assigned to triterpenoid 7 (Figure 2), which was named tricholidic acid C. Biosynthetic considerations, the cooccurrence in the same fruiting bodies, and the negative sign of the Cotton effect at about 220 nm suggested the absolute configuration of 7 to be the same as that of tricholidic acids 5 and 6 ( Figure 2). Compound 8 (Figure 2) was identified as tricholomenyn C by the identity of the physicochemical and NMR data within the literature [24] and comparison with an authentic sample.

Discussion
The chemical constituents of the fruiting bodies of T. ustaloides were investigated for the first time. Six rare representative metabolites of different biogenesis were isolated from this mushroom, namely, dimeric sesquiterpenoid saponaceolides 1 and 2, triterpenoids 5-7 with a modified lanostane structure, and tricholomenyn C (8) of mixed biogenesis. Saponaceolides J (1) and F (2) were previously isolated only once from nature, namely, from T. terreum [29] and from T. saponaceum [20,30], respectively. Thus, with the occurrence of 1 and 2 in T. ustaloides, examples of terpenoids with the saponaceolide skeleton have been found as characteristic metabolites of Tricholoma species placed in the sections Contextocutis (= sect. Rigida), Terrea, Atrosquamosa, and Genuina [4,7,17]. Tricholidic acid (5) was previously isolated only once, namely, from a Tricholoma species collected in Japan [31], which is probably identifiable as T. albobrunneum [11]; instead, tricholidic acids B (6) and C (7) are novel triterpenoids, isolated for the first time from T. ustaloides in this study. Interestingly, from a chemotaxonomic point of view, both T. albobrunneum and T. ustaloides belong to the section Genuina of Tricholomas [4,7]. This section also includes the species T. imbricatum, which produces the degraded lanostane triterpenoids tricholimbrins A and B, which are structurally related to tricholidic acids 5-7 [35]. Finally, this is the second finding in nature of tricholomenyn C (8), which was isolated for the first time by us from T. acerbum [24], which belongs to the section Megatricholoma [4,7].
The compounds 1, 2, 5-8 belong to families which, although distributed in different sections of Tricholomas, appear to be characteristic metabolites of the genus. In fact, there are no other examples of these types which have been isolated from mushrooms or other living organisms.
The cytotoxicity against five human cancer cell lines, determined by an MTS assay [34], revealed that saponaceolides J (1) and F (2) possessed high cytotoxicity (Table 2), confirming a biological property which appears to be common to the members of this family of secondary metabolites [18][19][20]. Preliminary results indicated that the cytotoxicity might be attributed to the presence of an α-alkylidene-γ-butyrolactone unit capable of undergoing a Michael reaction with biological nucleophiles such as L-cysteine or thiol-containing enzymes [36]. However, synthetic studies showed that a simplified structure, mimicking the right hand of the saponaceolide molecules and devoid of the dissymmetric tricyclospiroketal moiety, did not match the pattern of antitumor activities of the entire molecules. These results pointed out the need of the entire skeleton of saponaceolides for maintaining the specificity and potency of the cytotoxic activity [37]. Moreover, the comparison of the IC 50 values of 1-4 (Table 2) suggested that an OH group on the γ-lactone ring of saponaceolides (2 vs . 1 and 4 vs. 3), as well as the introduction of a double bond between C9 and C10 (1/2 vs. 3/4), increased the cytotoxicity.

Extraction and Isolation
Fresh fruiting bodies were brought in jute bags to the laboratory about 2 h after the collection and quickly frozen at −20 • C. Subsequently, they were roughly chopped and soaked three times with EtOAc (1.5 L each time) at −20 • C for 2 h each time. The extracts were put together and evaporated under reduced pressure in a water bath at <35 • C until 1/10 the original volume. Hexane (400 mL) was added, and the solution was evaporated to half volume; an additional volume of hexane was added, and the procedure was repeated three more times. Eventually, the solution was taken to dryness to give a brown oily residue (4.18 g). The residue was partitioned between MeOH-H 2 O, 90:10 (200 mL), and hexane (30 mL) four times to give a crude hexane extract (1.2 g) containing triglycerides (NMR) and other non-polar compounds. After methanol removal by evaporation, the aqueous phase was diluted with brine (60 mL) and extracted exhaustively with EtOAc. After drying (Na 2 SO 4 + MgSO 4 ), evaporation of the organic phase under reduced pressure afforded a dense oily residue (2.8 g). This mixture was subjected to silica gel CC (300 g). Elution with a CH 2 Cl 2 -Me 2 CO gradient (10:1-1:1), followed by MeOH, afforded seven main fractions (I-VII). Preliminary TLC analysis of these fractions on silica gel and reversed phase plates revealed that I, VI, and VII contained complex mixtures of compounds including those occurring in less complex fractions II-V. Therefore, I, VI, and VII were not analyzed. Fraction II (130 mg) was separated by a semipreparative MPLC silica gel column (16 g). Elution with a CH 2 Cl 2 -Me 2 CO gradient (40:1-5:1) afforded unidentified compound A1 (51 mg) and saponaceolide J (1, 6.7 mg). Fraction III (180.3 mg) was separated by silica gel CC (19 g). Elution with CH 2 Cl 2 -Me 2 CO (10:1) gave subfractions III-1-III-4. Subfraction III-2 contained tricholidic acid C (7, 3.  Table 1; IR (KBr):   1 (q, C13)  identical with those reported in the literature [24] and w an authentic sample. See supplementary material.

Cytotoxicity (MTS) Assays
The MTS assay protocol is based on the reduction of the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] yellow tetrazolium compound by NAD(P)H-dependent dehydrogenase enzymes in metabolically active cells to a purple formazan salt that is soluble in cell culture media [34]. The formazan salt has a maximum absorbance in the UV spectrum at 490 nm. The measure of the absorbance can be directly related to the number of viable (living) cells.
Five human cancer cell lines, myeloid leukemia HL-60, lung cancer A-549, hepatocellular cancer HepG2, renal cancer Caki-1, and breast cancer MCF-7, obtained from ATCC, were used in the MTS test. The cells were cultured in RPMI-1640 or DMEM/F-12 medium (Euroclone S.p.A, Milan, Italy) supplemented with 10% fetal bovine serum (FBS), 100 µg/mL penicillin/streptomycin and 2 mM L-glutamine (Life Technologies, Milan, Italy) at 37 • C under a humidified atmosphere added of 5% CO 2 , changing the liquid growth medium whenever needed. When a cell culture reached 75% confluence, a small amount of trypsin was added to the medium to separate the cells from the flask; after 3 min of incubation at 37 • C, 1 mL of FBS was added to stop the action of trypsin and to avoid degradation of cell membrane. The cell-containing medium was then transferred into a cell strainer and centrifuged (ALC 4232 Centrifuge) at 1000 rpm for 10 min. The resulting pellet was resuspended in the growth medium (1 mL), and the cells were separated using an automatic pipette and counted using a counting chamber and trypan blue as dye. Separated cells were seeded into each well of 96-well flat-bottom microplates (Cellstar, Greiner bio-one, Kremsmunster, Austria) at a density of 3 × 10 5 cells/mL of growth medium in each well. After 2 h of incubation the medium was replaced with 100 µL of test medium (RPMI 1640, added with 0.005% L-glutamine, penicillin, and streptomycin), and the microplates were left in the incubator for 24 h. For the IC 50 determination, six concentrations ranging from 0.1 to 100 µM in DMSO were prepared for each sample, including the cisplatin used as the cytotoxic reference compound. The medium in the wells was replaced with a solution (100 µL) of increasing sample concentration. Three replicates were performed for each sample dilution. The microplates were then incubated for 24 h; subsequently, the sample containing medium was replaced with fresh test medium (100 µL) and 20 µL of MTS tetrazolium reagent (CellTiter 96 ® -AQueous One Solution Cell Proliferation Assay, Promega Italia S.R.L., Milan, Italy). After 2 h incubation the absorbance at 490 nm was measured using a plate reader (BioRAD Model 550 Microplaate Reader). After each sample treatment, cell viability was detected, and a cell growth curve (mean absorbance vs. sample concentration) was graphed. IC 50 values, expressed in µM (Table 2), were calculated by probit analysis (p < 0.05, χ 2 test).

Conclusions
The first chemical analysis of the mushroom Tricholoma ustaloides led to the isolation and structure elucidation of two novel lanostane triterpenoids, named tricholidic acids B (6) and C (7). In addition, known saponaceolides J (1) and F (2), tricholidic acid (5), and tricholomenyn C (8), together with triglycerides, a mixture of free fatty acids, and five unidentified compounds A1-A5, were isolated from an EtOAc extract of the fruiting bodies. This is the second isolation of metabolites 1, 2, 5, and 8 from nature. Moreover, saponaceolides J (1) and F (2) showed high cytotoxicity (IC 50 values ≤ 10 µM) in an MTS assay against a panel of human cancer cells.
Our findings have confirmed that Tricholoma species are rich sources of novel compounds, most of which have unique chemical structures and interesting bioactivities. Therefore, we will further investigate the chemical contents of Tricholoma species that grow in the wild in Italy. Regarding the metabolites of T. ustaloides, we aim to establish the structures of the still unidentified compounds, A1-A5, that have been isolated in this study.